One-step generation of tumor models by base editor multiplexing in adult stem cell-derived organoids

Optimization of CRISPR/Cas9-mediated genome engineering has resulted in base editors that hold promise for mutation repair and disease modeling. Here, we demonstrate the application of base editors for the generation of complex tumor models in human ASC-derived organoids. First we show efficacy of cytosine and adenine base editors in modeling CTNNB1 hot-spot mutations in hepatocyte organoids. Next, we use C > T base editors to insert nonsense mutations in PTEN in endometrial organoids and demonstrate tumorigenicity even in the heterozygous state. Moreover, drug sensitivity assays on organoids harboring either PTEN or PTEN and PIK3CA mutations reveal the mechanism underlying the initial stages of endometrial tumorigenesis. To further increase the scope of base editing we combine SpCas9 and SaCas9 for simultaneous C > T and A > G editing at individual target sites. Finally, we show that base editor multiplexing allow modeling of colorectal tumorigenesis in a single step by simultaneously transfecting sgRNAs targeting five cancer genes.


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Life sciences study design
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Any available donated intestinal, endometrium and hepatocyte tissue was used to derive organoid lines and stored in biobanks in liquid nitrogen. Organoid lines in this study were selected from these biobanks based on the ease of culturing. Growth speed and splitting ratios may vary depending on the donor.
No Pre-determined sample sizes were chosen in case of editing efficiency experiments. Editing efficiencies were always determined based on the genotype of individual clones of at least n=2 transfection events. Numbers depend on the survivability of the clones after electroporation. For each experiment we grew out at least 16 individual clonal organoid lines (n) to get a sense of editing efficiency by sanger sequencing. in multiplexing experiments we drastically increased the sample size to 96 (n=96) to get a better sense of co-mutation in our dataset. for WGS at least n=3 individual samples were used for off-target analysis.
we did not exclude any data in this study To verify reproducibility of our strategy we use organoids derived from three distinct human tissues. Editing efficiencies were always Reporting for specific materials, systems and methods We require information from authors about some types of materials, experimental systems and methods used in many studies. Here, indicate whether each material, system or method listed is relevant to your study. If you are not sure if a list item applies to your research, read the appropriate section before selecting a response. Validation determined based on the genotype of individual clones of at least n=2 transfection events. sgRNA's were tested prior to multiplexing experiments and effectivity was always reproducible.
All of our studies are based on the genotype-phenotype relationships. As the mutated organoids in our study look vastly different compared to wild types, randomization is not sensible.
All of our studies are based on the genotype-phenotype relationships. As the mutated organoids in our study look vastly different compared to wild types, blinding is not sensible. B) Goat anti-rabbit Alexa Fluor 647, Invitrogen A21245 Anti-Rabbit secondary antibodies are affinity-purified antibodies with well-characterized specificity for rabbit immunoglobulins and are useful in the detection, sorting or purification of its specified target. Secondary antibodies offer increased versatility enabling users to use many detection systems (e.g. HRP, AP, fluorescence). They can also provide greater sensitivity through signal amplification as multiple secondary antibodies can bind to a single primary antibody. Most commonly, secondary antibodies are generated by immunizing the host animal with a pooled population of immunoglobulins from the target species and can be further purified and modified (i.e. immunoaffinity chromatography, antibody fragmentation, label conjugation, etc.) to generate highly specific reagents.